Vaccine for treating nicotine addiction

ABSTRACT

The present invention provides active or passive forms of immunotherapy against the metabolites of nicotine. For example, there is provided a vaccine comprising a metabolite of nicotine, and in particular those vaccines that comprise cotinine, which is used for immunotherapy of nicotine addiction.

The present invention relates to novel vaccines and pharmaceuticalpreparations for the treatment and prevention of nicotine addiction. Inparticular it relates to vaccines comprising one of the metabolites ofnicotine, or pharmaceutical preparations comprising ligands capable ofbinding to a metabolite of nicotine. The vaccines of the presentinvention are capable of inducing antibodies which abrogate the effectsof the metabolite of nicotine in the body, thereby facilitating smokingcontrol. Also provided by the present invention are novel immunogenscomprising a metabolite of nicotine and their use in the formulation ofvaccines for smoking control. Methods of treatment of individualswanting to reduce or quit smoking by administration of the vaccines orpharmaceutical preparations of the present invention, are also provided.

Smoking has an adverse effect on health and consequently smokers oftentry to quit the habit. However, the addictive nature of nicotine and theavailability of cigarettes add to the continued dependence on nicotineand the high failure rate of those trying to quit. Withdrawal symptomsare unpleasant and are usually relieved by further smoking.

One of the major causes of failure to quit smoking is the physicaladdiction to nicotine. Following administration, nicotine stimulatesmany receptors in the brain, causing release of dopamine, and thereafternicotine undergoes extensive biotransformation via a number of metabolicpathways (Kyerematen (1991) Dmg Metabolism Reviews 23(1&2), 3-41) intomany different metabolites, examples of which include cotinine, nicotine1′-N-oxide, nicotine glucuronide, nornicotine (Nicotine Safety andToxicity, Benowitz 1998), cotinine N-oxide, norcotinine,trans-3′-hydroxycotinine and cotinine glucuronide.

Cotinine has been shown to be a major metabolite of nicotine and a studyby Benowitz, (Clin Pharmacol Ther (1983) 34(5), 604-611) estimated that86% of systemically absorbed nicotine is metabolized to cotinine inhumans. Cotinie has also been shown (Dwoskin et al, The Journal ofPharmacology and Experimental Therapeutics (1999), 288(2), 905-911) tobe the most abundant metabolite in rat brain after peripheral nicotineadministration. Studies also suggest that cotinine has psychologicactivity and that it can antagonise the effects of nicotine in-vivo inhumans and animals. (Hatsukami et al. Psychopharmacology (1998)135:141-150).

Many therapies for nicotine addiction have been developed and used,which focus on the activities of nicotine itself. These therapies haveeither been in the form of nicotine replacement therapy, such asnicotine containing chewing gum or skin patches, or in the abrogation ofthe effects of nicotine by vaccination as described in WO 98/124216(Immulogic Pharmaceuticals Inc) and WO 00/33229 (Nabi). In contrast tothese references, the current invention involves the targeting of themetabolites of nicotine.

It has been found, surprisingly, that the targeting of the metabolitesof nicotine, by immunotherapy, facilitates smoking control. Inparticular, one aspect of the present invention involves theimmunotherapeutic alteration of the biological effects of themetabolites of nicotine which themselves are antagonists of nicotine.The result of this type of immunotherapy in an individual attempting toquit smoking is the reduction, prevention or alteration of smokingwithdrawal symptoms, or the alteration of the positive effects ofnicotine intake, thereby increasing the chances of success of theindividual quitting smoking. The metabolite of nicotine that isparticularly preferred within the context of the present invention iscotinine.

Without wishing to be bound by theory, an immune reaction in a subjectagainst a cotinine metabolite can surpisingly reduce the dampeningeffect the cotinine metabolite has on the nicotine neurological effect,thereby resulting in the subject craving a lesser amount of nicotine.

The present invention, therefore, provides active or passive forms ofimmunotherapy against the metabolites of nicotine. For example, there isprovided a vaccine comprising a metabolite of nicotine, and inparticular those vaccines that comprise cotinine, which is used forimmunotherapy of nicotine addiction.

Also provided are immunogens for use in the vaccines of the presentinvention comprising a metabolite of nicotine, conjugated to a carriermolecule that is capable of converting the poorly immunogenic metaboliteinto an effective immunogen.

Accordingly, there is provided an immunogen comprising a metabolite ofnicotine, and a carrier molecule that comprises T-helper epitopes, suchas a protein, for use in the immunotherapy of nicotine addiction.Preferred immunogens of the present invention comprise cotinineconjugated to a protein carrier molecule.

For example anti-cotinine antibodies were purchased from AmericanResearch Products Inc (ARP), 489 Common St. #B, Belmont, Mass.,02178-4455.

Anti-cotinine antibodies are also available from Cortex Biochem Inc.,1933 Davis St., #321, San Leandro, Ca, 94577 and AdvancedBiotechnologies Ltd., Mole Business Park 3, #7 Randalls Road,Leatherhead, Surrey, KT22 7B.

There is also provided by the present invention, use of the metabolitesof nicotine, and especially cotinine, in the manufacture of a medicamentfor the treatment of nicotine addiction.

Also provided by the present invention is the passive therapy ofnicotine addiction. Accordingly, pharmaceutical preparations areprovided for administration which comprise a ligand capable of bindingto, and reducing the activity of, a metabolite of nicotine. In thisaspect of the present invention the preferred ligands are polyclonal ormonoclonal antibodies. Again, the preferred metabolite of nicotine forpassive immunotherapy is cotinine.

The term “antibody” herein is used to refer to a molecule having auseful antigen binding specificity. Those skilled in the art willreadily appreciate that this term may also cover polypeptides which arefragments of or derivatives of antibodies yet which can show the same ora closely similar functionality. Such antibody fragments or derivativesare intended to be encompassed by the term antibody as used herein.

Accordingly, in a related aspect of the present invention are ligandscapable of binding to, and reducing the activity of cotinine. Theimmunogens of the present invention may also be used for the generationof monoclonal antibody hybridomas (using known techniques e.g. Köhlerand Milstein, Nature, 1975, 256, p495), humanised monoclonal antibodiesor CDR grafted monoclonals, by techniques known in the art. The use ofthe immunogens described herein in the manufacture of polyclonal ormonoclonal antibodies for the treatment of nicotine addiction is alsoprovided by the present invention.

Novel immunogens are provided which comprise the conjugation of ametabolite of nicotine to a T-helper epitope containing protein carrier.

Cotinine is shown by the following structure:

Immunogens of the present invention are made by the linking the proteincarrier to any position on the cotinine molecule provided that thelinker does not join at the 2′ (carbonyl) position.

Particularly preferred cotinine based immunogens of the presentinvention are shown in the following formula:

wherein a protein carrier is covalently coupled to the cotinine moleculeat any of the positions 1, 2, 5, 6, or 4′.

Specific linkers and the corresponding linker chemistry is shown in WO98/124216 (Immulogic Pharmaceutical Corporation) WO 00/33229 (Nabi) andWO 99/61054 (Independent Pharmaceutica AB), all of which areincorporated by reference hereto.

Covalent coupling of the peptide of the metabolite to the immunogeniccarrier can be carried out in a manner well known in the art. Thus, forexample, for direct covalent coupling it is possible to utilise acarbodiimide, glutaraldehyde or (N-[γ-maleimidobutyryloxy]) succinimideester, utilising common commercially available heterobifunctionallinkers such as CDAP and SPDP (using manufacturers instructions). Afterthe coupling reaction, the immunogen can easily be isolated and purifiedby means of a dialysis method, a gel filtration method, a fractionationmethod etc.

The types of carriers used in the immunogens of the present inventionwill be readily known to the man skilled in the art. The function of thecarrier is to provide cytokine help in order to help induce an immuneresponse against the metabolite of nicotine. A non-exhaustive List ofcarriers which may be used in the present invention include: Keyholelimpet Haemocyanin (KLH), serum albumins such as bovine serum albumin(BSA), inactivated bacterial toxins such as tetanus or diptheria toxins(TT and DT), CRM197 or recombinant fragments thereof (for example,Domain 1 of Fragment C of TT, or the translocation domain of DT), or thepurified protein derivative of tuberculin (PPD). Alternatively thenicotine metabolites may be directly conjugated to liposome carriers,which may additionally comprise immunogens capable of providing T-cellhelp. Preferably the ratio of the number of nicotine metabolites to eachcarrier is in the order of 1:1 to 20:1, and preferably each carriershould carry between 3-15 nicotine metabolites.

In an embodiment of the invention a preferred carrier is Protein D fromHaemophilus influenzae. Protein D is an IgD-binding protein fromHaemophilus influenzae and has been patented by Forsgren (WO 91/18926,granted EP 0 594 610 B1). In some circumstances, for example inrecombinant immunogen expression systems it may be desirable to usefragments of protein D, for example Protein D ⅓ (comprising theN-terminal 100-110 amino acids of protein D (GB 9717953.5)).

Alternatively, the carrier molecule may consist of T-helper epitopes, inthe absence of other non-helper amino acid sequences (WO 99/67293), orthe metabolite of nicotine may be formed into immunogenic matrices (WO00/33229).

The present invention, therefore, provides the use of the metabolites ofnicotine in the manufacture of vaccines for the prophylaxis or therapyof nicotine addiction. Accordingly, the immunogens of the presentinvention are provided for use in medicine, and in the medical treatmentor prophylaxis of nicotine addiction. Accordingly, there is provided amethod of treatment of nicotine addiction comprising the administrationto a patient suffering from or susceptible to nicotine addiction, of avaccine or medicament of the present invention.

Vaccines of the present invention, may advantageously also include anadjuvant. Suitable adjuvants for vaccines of the present inventioncomprise those adjuvants that are capable of enhancing the antibodyresponses against the metabolite of nicotine. Adjuvants are well knownin the art (Vaccine Design—The Subunit and Adjuvant Approach, 1995,Pharmaceutical Biotechnology, Volume 6, Eds. Powell, M. F., and Newman,M. J., Plenum Press, New York and London, ISBN 0-306-44867-X). Preferredadjuvants for use with immunogens of the present invention includealuminium or calcium salts (for example hydroxide or phosphate salts).Other adjuvants include saponin adjuvants such as QS21 (U.S. Pat. No.5,057,540) and 3D-MPL (GB 2220 211).

The vaccines of the present invention will be generally administered forboth priming and boosting doses. It is expected that the boosting doseswill be adequately spaced, or preferably given yearly or at such timeswhere the levels of circulating antibody fall below a desired level.

In a further aspect of the present invention there is provided a vaccineas herein described for use in medicine.

The vaccine preparation of the present invention may be used to treat anindividual addicted to nicotine, by means of administering said vaccinevia systemic or mucosal route. These administrations may includeinjection via the intramuscular, intraperitoneal, intradermal orsubcutaneous routes; or via mucosal administration to theoral/alimentary, respiratory, genitourinary tracts. A route ofadministration via the transdermal route is, for example by vaccinedelivery skin patches.

The amount of protein in each vaccine dose is selected as an amountwhich induces an immunoprotective response without significant, adverseside effects in typical vaccines. Such amount will vary depending uponwhich specific immunogen is employed and how it is presented. Generally,it is expected that each dose will comprise 1-1000 μg of protein,preferably 1-500 μg, preferably 1-100 μg, of which 1 to 50 μg is themost preferable range. An optimal amount for a particular vaccine can beascertained by standard studies involving observation of appropriateimmune responses in subjects. Following an initial vaccination, subjectsmay receive one or several booster immunisations adequately spaced.

Pharmaceutical compositions comprising the ligands, described above,also form an aspect of the present invention. Also provided are the useof the ligands in medicine, and in the manufacture of medicaments forthe treatment of nicotine addiction.

Vaccine preparation is generally described in New Trends andDevelopments in Vaccines, edited by Voller et al., University ParkPress, Baltimore, Md., U.S.A. 1978. Conjugation of proteins tomacromolecules is disclosed by Likhite, U.S. Pat. No. 4,372,945 and byArmor et al., U.S. Pat. No. 4,474,757.

The present invention is illustrated by but not limited to the followingexamples.

1. Preparation of Cotinine-Protein Conjugates

1.1 Preparation of N-alkyl Pyridyl Cotinine Conjugate

The production of the following derivatised cotinine molecule wasperformed as described below.

The reaction was conducted under nitrogen, and all glassware waspredried in an oven at 150° C. for at least 12 hours prior to use.

(−)-Cotinine (200 mg, 1.1 mmol) was purchased commercially from TorontoResearch Chemicals, (www.trc-canada.com) and dissolved in dry methanol(1 cm³, distilled from CaH₂) and cooled to 0° C. in an ice bath and6-bromohexanoic acid (244 mg, 1.25 mmol) in dry methanol was addeddropwise. The solution was then allowed to stir at room temperature for24 hours. The solvent was then removed in vacuo, and water (5 cm³) andCH₂Cl₂ (5 cm³) were then added to the residue. The aqueous phase waswashed with CH₂Cl₂ (6×5 cm³) (to remove excess bromoacid) until theaqueous phase only showed one baseline product by TLC (methanol). Theaqueous solution was then concentrated (freeze drier) to give theN-alkylpyridyl cotinine conjugate (90 mg, 27%) as a colourless oil,δ_(H) (270 MHz, CDCl₃) 8.84 (2H, d, J 6.3), 8.47 (1H, d, J 7.9), 8.09(1H, dd, J 6.3, 7.9), 5.03 (1H, m), 4.62 (2H, t, J 7.3), 2.72-2.54 (6H,m), 2.36 (2H, t, J 7.3), 2.09-1.88 (3H, m), 1.68-1.56 (2H, m), 1.41-1.29(2H, m); m/z (EI) 291 [M⁺, 3%], 176 [M-((CH₂)₅CO₂H), 42%].

Conjugating carboxyl-group-containing molecules to proteins.

Carboxyl groups can be conjugated to amino-groups on proteins usingeither an active ester intermediate (for example making aN-hydroxysuccinimide ester), or by activating the carboxyl group in situwith carbodiimide.

Carbodiimide:

When linking to proteins the water soluble carbodiimide is usually used.This is referred to as EDC or EDAC. Other carbodiimides exist, but aregenerally for organic synthesis.

The hapten is dissolved in water so that there will be a 20-100 foldmolar excess over the protein. The pH is adjusted so that the carboxylgroup is in the protonated form: this generally requires bringing the pHto below 7, and ideally to about pH 5. At higher pHs the reaction mightalso work however a higher ratio of hapten to protein may be required inorder to get the reaction to work.

A 10-fold molar excess (over the carboxyl group) of soluble carbodiimide1-ethyl-3-(3-dinethylaminopropyl)carbodiimide (EDC) is then added. Thiscan be added either as a powder, or as a very fresh solution in water.This is left to incubate at room temperature for 3-5 minutes.

The activated hapten-EDC mixture is then added to the protein solutionin 100 mM phosphate buffer at pH 8. This buffer system prevents theresidual carbodiimide activating carboxyl groups on the protein.

Leave for several hours (5-8) and dialyse against phosphate bufferedsaline to remove any unreacted derivatised cotinine.

Hydroxysuccinimide Esters

Instead of activating the carboxyl group in situ, for small organicmolecules the activation can be done beforehand.

In this case N-hydroxysuccinimide is conjugated to the carboxyl group ofthe hapten, generally using an organic-soluble carbodiimide such asdicyclohexylcarbodiimide (DCC). The resulting N-hydroxysuccinimide esterwill conjugate automatically to amino-groups on proteins:

The requirements are that the activated hapten is soluble in water, andthat the protein is at a pH between 7.5 and 8.5.

If the N-hydroxysuccinimide esters are not soluble directly in water,the activated hapten can be dissolved in dimethyl formamide or dimethylsulphoxide or ethanol, and then a small amount of this organic solutionadded to the solution of protein in 50 mM phosphate buffer pH 7.5-8. Itis important that the organic solution is fairly fresh: the presence oftrace amounts of water in these solutions will slowly hydrolyse theactive ester. In the subsequent conjugation reaction no more than a10-20% by volume of organic solvent should be added to the proteinsolution to prevent the protein from precipitating.

An alternative method is to make the sulfo-N-hydroxysuccinimide ester.In this case there is a sulphate group on the N-hydroxysuccinimide,enhancing the water—solubility.

Generally a 10- or 20-fold molar excess of the activated ester is addedto the protein solution in 20-50 mM phosphate buffer pH 7.5-8. A proteinconcentration of around 1 mg/ml is generally used. Allow the reaction toproceed for 5 hours at least, and then dialyse against phosphatebuffered saline to remove any unreacted derivatised cotinine. Dialysisshould be at 4° C., and against at least two changes of buffer, and eachdialysis should last at least 3 hours.

Other methods of conjugating carboxyl groups to proteins have beendescribed, eg by adding sulfo-N-hydroxysuccinimide to the EDC reactionto help it (in situ formation of a sulfo-NHS active ester intermediate)or using HATU.

1.2 Preparation of Thiol Conjugate of Cotinine

The production of the following derivatised cotinine molecule wasperformed as described below.

The reaction was conducted under nitrogen, and all glasswre was predriedin an oven at 150° C. for at least 12 hours prior to use.

(±)-trans-4-Cotinine carboxylic acid (200 mg, 0.9 mmol),2-aminoethanethiol.HCl (103 mg, 0.9 mmol) and triethylamine (0.13 cm³,1.8 mmol) were dissolved in dry DMF (1.5 cm³). The mixture was cooled to0° C. in an ice bath, and EDCI.HCl (174 mg, 0.9 mmol) was then added.The reaction mixture was warmed to room temperature and stirred for afurther 12 hours. The solvent was removed in vacuo, and water (5 cm³)and CH₂Cl₂ (5 cm³) were added to the residue. The aqueous phase wasextracted with CH₂Cl₂ (4×5 cm³), and the combined organic extracts weredried over Na₂SO₄, filtered, and concentrated in vacuo. Purification byflash chromatography using 5% methanol/dichloromethane as eluent, gavethe thiol (136 mg, 54% yield) as a colourless oil, δ_(H) (400 MHz,CDCl₃) 8.58 (1 H, dd, J 2.0, 4.9, H-4), 8.50 (1H, d, J 2.0, H-2), 7.60(1H, dt, J 2.0, 7.8, H-6), 7.38 (1H, dd, J 4.9, 7.8, H-5), 6.77 (1H, brt, J 5.4, NH), 4.79 (1H, d, J 6.8, H-7), 3.51-3.35 (2H, m, H-9a, H-9b),2.92-2.68 (5H, m, H-10, H-13a, H-13b, H-14a, H-14b), 2.64 (3H, s, CH₃),1.32 (1H, t, J 8.3, SH); ν_(max) (thin film)/cm⁻¹3500-3100 (br NH), 2543(S—H), 1684 (C═O).

The derivatised cotinine was then conjugated via a thio-ether bond toOvalbumin as the protein carrier using conventional maleimide chemistry.Heterobifunctional reagents such as GMBS(gamma-maleimidobutyryloxy-succinimide ester) react with amino groups onproteins via the hydroxysuccinimide ester, resulting inmaleimide-modified protein, which can then react with thiols.

Manufacturers' recommendations should be followed, but the followingconditions were used. GMBS is dissolved in ethanol or dimethylformamideat 1 mg/ml, dissolve protein (OVA) in phosphate buffer or PBS pH 7.5-8.Add a 10-20 fold molar excess of GMBS to the protein. A 10-fold excesstends to give about 5 maleimide groups per ovalbumin molecule. A 20-foldexcess gives about 10 maleimide groups per ovalbumin. Allow to react for2-3 hours. Dialyse against a large volume (1 l) of phosphate buffer ofPBS pH 7.5. After 34 hours of dialysis add your thiolated reagent to themodified protein. The thiolated reagent should be added in excess overthe maleimide groups, such as a 20-fold excess. Allow to react for >8hours. Dialyse for several hours to remove unbound hapten.

1.3 Preparation of the 6-amino Cotinine Conjugate

The production of the following derivatised cotinine molecule wasperformed as described below.

Acrylic acid (0.04 cm³, 0.6 mmol) was added to a solution of 6-aminocotinine (100 mg, 0.5 mmol) in toluene (1 cm³) and reflux, and heatingwas continued for one hour. During this time an oil separated out. Thereaction mixture was cooled to room temperature, and the solvent removedin vacuo. Water (5 cm³) and dichloromethane (5 cm³) was added, and theaqueous phase was separated extracted with further portions ofdichloromethane (3×5 cm³). The combined organic extracts were combinedand the solvent was removed in vacuo. Purification by flashchromatography using 2% methanol/dichloromethane as eluent, gave the6-amino cotinine conjugate (12 mg, 9%) as a colourless oil, δ_(H) (270MHz, CDCl₃) 8.66 (1H, br s, NH), 8.36 (1H, d, J 8.5, H-5), 8.21 (1H, d,J 2.2, H-2), 8.21 (1H, dd, J 2.2, 8.6, H-4), 6.51 (1H, dd, J 1.5, 17.0,H-14a), 6.36 (1H, dd, J 9.8, 17.0, H-13), 5.84 (1H, dd, J 1.5, 9.8,H-14b), 4.54 (1H, m), 2.70 (3H, s, CH₃), 2.67-2.46 (3H, m), 1.97-1.79(1H m).

The acrylamide group of the 6 amino cotinine derivative can be easilyconverted to a free thiol group by reacting it with thioacetic acid orpotassium thioacetate. The free thiol group is then generated bycleaving the thioacteate using either potassium carbonate/methanol orsodium methoxide in methanol. The derivatised cotinine was thenconjugated via a thio-ether bond to Ovalbumin as the protein carrierusing conventional thiol chemistry.

The heterobifunctional reagent SPDP (N-succinimidyl3,2-pyridyldithiopropionate) is reacted with the amino groups on theprotein, to form protein derivatised with dithiopyridyl. Thedithiopyridyl group reacts with thiol groups at pH above 5 forming adisulphide bond (below pH 7 thiols do not react readily with otherthiols, so between pH 5 and 7 the reaction favours formation ofdisulphide bonds between the thiol-bearing hapten and the protein).

1.4 Formulation of Vaccines

The products of 1.1, 1.2 and 1.3 are formulated into vaccines byadsorbtion onto aluminium hydroxide salt purchased from Superfos.

1. A vaccine for use in the treatment of nicotine addiction ofindividuals wishing to quit smoking, comprising a metabolite of nicotineconjugated to a carrier comprising T-cell epitopes, and an adjuvant. 2.A vaccine according to claim 1, wherein the carrier is a protein.
 3. Avaccine according to claims 1 or 2, wherein a linker bridges themetabolite of nicotine and the carrier.
 4. A vaccine according to any ofclaims 1 to 3, wherein the metabolite of nicotine is cotinine.
 5. Avaccine according to claim 4, wherein the cotinine is conjugated to thecarrier via the 1, 2, 5, 6 or 4′ position.
 6. A vaccine according to anyof claims 1 to 5, for administration via the systemic or mucosal route.7. A method of treatment of nicotine addiction comprising theadministration to a patient suffering from or susceptible to nicotineaddiction, who wishes to quit smoking, of a vaccine according to any ofclaims 1 to
 6. 8. The use of a composition comprising a metabolite ofnicotine conjugated to a carrier comprising T cell epitopes in themanufacture of a medicament for the treatment of nicotine addiction ofindividuals wishing to quit smoking.
 9. A process for making a vaccinecomprising the steps of conjugating cotinine via the 1, 2, 5, 6 or 4′position to a carrier comprising T-cell epitopes, optionally via alinker, and adding an adjuvant to the resulting conjugate.